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Experiment 1: Estimation of Total Serum Cholesterol by Zak and Henlys Method

2014, 201428982, 201435733


Group 3, Biochemistry 35.1, MEG
Instructor: Ms. Ciara Christianne Lim
Date submitted: January 30, 2017 Monday

I. Abstract
Cholesterol, a steroid alcohol, regulates the fluidity of plasma membrane so right amount of
cholesterol in the body must be maintained because too much or too low amount of it may
cause different types of diseases. This study aims to quantify cholesterol level of serum
samples and compare it with clinically established cholesterol level value. To quantify the
amount of cholesterol in serum sample, a standard calibration curve was obtained by
preparing different concentrations of cholesterol and measuring its absorbance known as
Zak and Henlys method. From the equation obtained, it was found that the serum 1 and
serum 2 contains 0.921 and 0.821 mg/dL cholesterol, respectively.

II. Keywords cholesterol


III. Introduction artheroscelerosis and coronary heart
Cholesterol is a steroid alcohol found in disease (Carmena, MD, Duriez, PhD, &
abundance in animals. It has a branched Fruchart, PhD, 2004). Low levels of
aliphatic side-chain and OH group cholesterol in the blood have been
attached to the C17 and C3 of the steroid connected to an increased risk for liver
nucleus, making it an amphiphatic and pancreatic cancer, hepatic cirrhosis,
molecule (Voet & Voet, 2011). Its structure suicide and alcohol dependency syndrome
is shown on figure 1. (Neaton, PhD, Blackburn, MD, Jacobs, PhD,
& Lee, MD, 1992). It is therefore important
to be able to quantify the total serum
cholesterol in order for one to assess
whether or not their blood cholesterol is
within safe levels.

The objectives of the experiment are to


prepare test serum samples from blood, to
generate a standard calibration curve for
cholesterol, to quantify the cholesterol
Figure 1. The structure of cholesterol (Voet & Voet,
2011) level of the test serum samples and to
assess and compare the cholesterol level
Cholesterol functions in the regulation of with clinically established values.
the fluidity of animal plasma membranes.
It is also the precursor to steroid
IV. Experimental
hormones, which are important regulators
of several physiological functions, such as
the development of secondary sex A Blood Serum Preparation
characteristics (Voet & Voet, 2011).
Ten milliliters of whole blood was
Cholesterol, however, is widely known for incubated at room temperature for 15-
the health hazards attributed to it. High 30 minutes. Coagulated masses/ Blood
levels of cholesterol in the blood, a clots were then removed via
condition called hypercholesterolemia, centrifugation at 1500 xg for 10
increases the risk for the development of minutes. *The supernatant (serum)
cardiovascular diseases such as was transferred into a falcon tube and
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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any turbid samples were further
centrifuged and had their supernatant C Total Serum Cholesterol Measurement
collected. Aliquots of 0.5 mL were
placed in microcentrifuge tubes and A 0.05 mL aliquot of test serum sample
stored at -80C. was mixed with 4.95 mL ferric chloride
reagent to form a 1% v/v serum
B Generation of the Cholesterol Standard solution. This was done in duplicate.
Calibration Curve The solutions were allowed to stand for
15 minutes, with occasional mixing.
//The cholesterol stock was prepared
by dissolving enough cholesterol in (Charles, pasabi na lang ditto na inulit
chloroform to reach 1mg/mL natin yung ginawa. Hehe. Di ko
concentration. The ferric chloride maformulate ng maayos kung ano
solution was prepared by dissolving sasabihin e hahaha. Salamat)
enough of Kilianis reagent with glacial Solutions which had precipitation were
acetic acid to form a 1% v/v solution.// centrifuged at 2000 xg for 15 minutes,
**this can be replaced by: and had their supernatant collected.
mixtures of the 1mg/ml cholesterol Three milliliters of concentrated
stock and 1% v/v ferric chloride sulfuric acid were added to each of the
solutions, with volumes shown on solutions. The solutions were incubated
***Im not sure which would be better at room temperature for 30 minutes.
though The absorbances of the solutions were
measured at 560 nm.
Mixtures of the cholesterol stock and *** copy-paste lang to from the
ferric chloride solution, with volumes previous part. Tell me if kailangan kong
shown on Table 1, were allowed to ireword for this part
stand at room temperature for 15
minutes. The cholesterol level (in mg/dL) of the
test sample was determined from the
Table 1 The volumes of the cholesterol stock
standard calibration curve *previously
and ferric chloride solution mixed for the
generation of the standard curve constructed.

Std Cholesterol Ferric


No. Stock (mL) Chloride (mL) V. Results and Discussion
1 0.00 5.00
2 0.05 4.95 Humans intake cholesterol-rich food in
3 0.10 4.90 their day to day lives. In fact, cholesterol
4 0.20 4.80 is needed for the structural components in
5 0.30 4.70 biological membranes and as a precursor
6 0.40 4.60 in the production of several steroids and
hormones as well as uses in hormone
Solutions which had precipitation were signalling. However, too much or too little
centrifuged at 2000 xg for 15 minutes, cholesterol in blood may lead to several
and had their supernatant collected. complications (Campbell & Farrell, 2010;
Three milliliters of concentrated Cox & Nelson, 2013).
sulfuric acid were added to each of the
solutions. The solutions were incubated
at room temperature for 30 minutes. Blood was obtained an hour prior to the
The absorbances of the solutions were experiment. This was done to ensure that
measured at 560 nm, and the standard the blood has sufficient time to coagulate.
calibration curve was constructed from The blood was allowed time to coagulate
this data. as the specimen needed for the
experiment was the blood serum. Blood
Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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serum is essentially blood plasma after it
undergoes adequate coagulation. Blood
serum is preferred over plasma as
Cholesterol Concentration ( mgdL )= Volume
Total Volume
(m
Stock Cholesterol Sol' n

Mixture ( mL

interference from proteins and ions are (Eq. 1)


minimized, both of which may affect the
results of biochemical analysis. The blood
serum sample was aliquoted to Sample computation for cholesterol
microcentrifuge tubes in 0.5mL volumes in concentration for Standard # 2,
order to prevent contamination and stored
in a refrigerator prior to use. The protein 0.05mL 1 mg 100 mL mg
fractional values of serum varies greatly =0.625
8 mL 1 mL 1 dL dL
as to what temperature it is stored.
Storage of serum at a temperature of 5 O Paa=lagay ng introduction dito about doon sa
10OC would ensure a negligible change in table. Salamat hehe.
protein fraction over a period of seven
Di ko gets Meh - Beah
days (Guder, et al., 2002; Kawai, 1973).
Guder, et al. (2002) reported that High Concentration vs. Absorbance Calibration Curve
Density Lipoproteins and Low Density
Lipoproteins would be stable for 7 days 0.990
within the temperature range of 4 O 8OC 0.790
and lesser at 20O 25OC. f(x) = 0.18x - 0.13
0.590
R = 0.89
Absorbance (560 nm)
After sample preparation, cholesterol 0.390
standard calibration curve was plotted.
0.190
The table below shows the absorbances
and their corresponding cholesterol -0.010
concentrations of the cholesterol standard 0.000 5.000
calibration curve as well as that of the [Cholesterol] in mg/dL
serum samples.

Table 1. Absorbances and their Corresponding Figure 1. Cholesterol Standard Calibration Curve.
Concentrations of the Cholesterol Standard
Calibration Curve and Serum Samples.
The obtained line equation (2) was
Average y=0.184 x 0.132
[Cholestero (Eq. 2)
Std. # Absorbance
l] (mg/dL)
(A)
1 0.000 0.000
2 0.625 -0.003 with an R value of 0.8871. Using
3 1.250 0.051 Equation 2, the total serum concentration
4 2.500 0.120 can be calculated.
5 3.750 0.539
6 5.000 0.917 Sample computation of total serum
Serum 1 0.921 0.038 cholesterol of Serum sample 1 using Eq. 2,
Serum 2 0.821 0.019
A=0.184[Cholesterol]0.132
Cholesterol concentration (in mg/dL) of
the standard calibration curve can be A +0.132
determined using Equation 1,
[ Cholesterol ] =
0.184

[ Cholesterol ] dilution factor=Total Serum Cholesterol

Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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mg 8.00 mL
0.921
dL 0.05 mL

mg
147.4
dL

Table 2. Total Serum Cholesterol of the Serum


Samples.
Total Serum
Sampl [Cholestero
Cholesterol
e# l] (mg/dL)
(mg/dL)
1 0.921 147.4
2 0.821 131.3

Total serum cholesterol (TSC) was Figure 3. Products of Cholesterol that underwent
estimated using the Zak and Henlys the LiebermannBurchard Reaction (Xiong, Wilson,
method. Zak and Henlys method involves & Pang, 2007).
a colorimetric reaction between
cholesterol and the ferric ions. Cholesterol Errors in this method of analysis may
reacts in the presence of sulphuric acid to arise due to several factors. First is due to
form 3, 5 cholestadiene (Figure 3) due to the interference of several compounds.
a dehydration reaction. In the presence of Several examples are bromide, bilirubin,
ferric (Fe3+) ion, 3, 5-cholestadiene tryptophan, and nitrate. It was studied
undergoes oxidation and is then that bromide would give a positive error as
sulphonated to form the red/purple- it enhances the color reaction with ferric
colored cholestapolyene sulphonic acid chloride resulting to a darker solution than
(reaction pathway shown in Figure 4). what is true. On the other hand, bilirubin is
Absence of ferric ions will result to a oxidised by a ferric chloride reaction to
solution that is green-colored, which is the form biliverdin that shows a minimum in
principle behind of the Liebermann spectral characteristics as well as lowered
Burchard reaction. The intensity of the absorptivity resulting to a negative error.
color is dependent upon the amount of Tryptophan may interfere with the analysis
cholesterol present in the serum given that glyoxal is present due to the
(Prasannan, Rajan, & Ramakrishnan, 2004; resulting Hopkins-Cole reaction but
Shivaraja Shankara, 2008). purification of acetic acid can be used to
prevent this. Interference of nitrate may
also be presumed however no further
information about how or why this
happens was available (Zak, 1965).

In blood, 30% of cholesterol exists in its


free form while the rest exists in its
esterified form. Regulation and monitoring
of cholesterol concentration in the body
especially of that in the blood is important
Figure 2. Structure of 3, 5-cholestadiene. in order to prevent several diseases.
(Retrieved from
http://www.pherobase.com/pherobase/gif/3,5-
Normal serum cholesterol levels for young
cholestadiene.GIF.) adults are usually in the range of 150-220
mg/dL though it is preferable to have a

Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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concentration below 200 mg/dL (Shivaraja substrate. This process is further
Shankara, 2008). enhanced due to the statins having an
affinity with the enzyme in the nanomolar
range versus that of the normal
Too little serum cholesterol
substrates affinity which in the millimolar
(Hypocholesterolemia) may result in
range. Furthermore, it also increases the
anemia, hyperthyroidism, hemolytic
activity of hepatic LDL receptors which
jaundice, etc. Hypocholesterolemia is
helps lead to the reduction of circulating
often a symptom of a serious underlying
LDL and its precursors (Sima & Stancu,
problem rather than a disease itself.
2001).
Currently, there is still no definable
cholesterol level below which clinically
significant hypocholesterolemia is VI. Conclusions and
diagnosed. Most agree on diagnosing Recommendations
hypocholesterolemia to those levels below
150 mg/dL. Others, to levels below 100
In this experiment, Zak and Henlys
mg/dL. Total serum cholesterol of the
method was used to create a standard
sample was found to be below 150 mg/dL
calibration curve to quantify the amount of
however this may not signify that the
cholesterol in serum sample. It is
sample donor is hypocholesterolemic as
important to monitor the amount of
standard TSC is also affected by gender
cholesterol in blood because too low or too
and race. Nevertheless, awareness of the
much amount of it causes several types of
condition may ensure a healthier lifestyle
diseases to arise like
and prevention of any serious diseases
hypercholesterolemia.
(Elmehdawi, 2008; Shivaraja Shankara,
2008). It is recommended to perform other
tests in measuring cholesterol level in
serum like enzymatic colorimetric method
On the other hand, too much serum that uses cholesteryl ester hydrolase
cholesterol or Hypercholesterolemia may (Simpson, 2012). Another possible
lead to uncontrollable Diabetes mellitus, technique that can be used is the alcohol-
atherosclerosis and coronary heart disease acetone technique (Zak, 1965).
(Shivaraja Shankara, 2008).
The ferric-chloride solution is
unstable at room temperature thus the
Table 3. Total Serum Cholesterol Standards color-formation capacity of the reagent is
(Dominiczak, Rifai, & Warnick, 2000). being lessen. This can be prevented by
adding Rosenthal, or using ferric
ammonium chloride dissolved in 80%
acetic acid. Bromide and tryptophan
causes interference by enhancing the
color of the complex that will yield to a
higher reading in the spectrometer. To
eliminate these interferences, serum must
Treatment of hypercholesterolemia be treated with ethanol and alkali and will
usually include the intake of drugs which be extracted with chloroform (Zak, 1965).
have statins. Statins are inhibitors of 3- Theoretically, Zak and Henleys
hydroxy-3-methylglutarylcoenzyme A method should be an effective way on
(HMG-CoA) reductase, an enzyme that measuring cholesterol in total serum
converts HMG-CoA into mevalonic acid samples but due to some errors that arise,
which is a cholesterol precursor. cholesterol level was not accurately
Additionally, they also change the measured.
conformation of the enzymes active site
rendering it unusable to the normal

Biochemistry 35.1: Estimation of Total Serum Cholesterol by Zak and Henlys Method
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Tanong lang, kailangan ba ilagay ko Prasannan, K. G., Rajan, R., &
equation, seru cholsetreol, at Ramakrishnan, S. (2004). Textbook
icompare sa standard? of Medical Biochemistry (Third ed.).
VII. References Hyderabad, India: Orient Blackswan.

Campbell, M. K., & Farrell, S. O. (2010). Shivaraja Shankara, Y. M. (2008).


Biochemistry (Seventh ed.). CA: Laboratory Manual for Practical
Cengage Learning: Brooks/Cole. Biochemistry (First ed.). New Delhi,
India: Jaypee Brothers Medical
Cox, M. M., & Nelson, D. L. (2013). Publishers.
Lehningers Principles in
Biochemistry (Sixth ed.). NY: W. H. Sima, A., & Stancu, C. (2001). Statins:
Freeman and Company. mechanism of action and effects.
Journal of Cellular and Molecular
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R. (2000). Handbook of Lipoprotein
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2012: http://www.acb.org.uk/Nat
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Xiong, Q., Wilson, W. K., & Pang, J. (2007).
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Y. S., Tpfer, G., Wisser, H., & Zawta, Sulfonation, Desaturation, and
B. (2002). Use of anticoagulants in Rearrangment of Cholesterol in
diagnostic laboratory investigations Acid. Lipids, 42(1), 8796.
& stability of blood, plasma and
serum samples. WHO/DIL/LAB/99.1 Zak, B. (1965). Total and Free Cholesterol.
Rev.2. Standard Methods of Clinical
Chemistry, 5, 7989.
Kawai, T. (1973). Clinical Aspects of The
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